Primary adult NSC neurosphere cultures were generated as previously described37 (link). Briefly, the lateral SVZs of five adult WT male mice were dissected per neurosphere culture and incubated in papain solution for 30 min at 37 °C with pipette dissociation every 10 min to obtain a single-cell suspension. Cells were then cultured in proliferating conditions (with addition of growth factors EFG and FGF) for 7 days at 37 °C to obtain primary neurospheres. To analyse cell differentiation, neurospheres were dissociated into single cells and plated on Poly-D-Lysine-coated glass coverslips in 24-well plates (50000 cells per well) in complete culture medium without proliferating factors for 1 to 5 days.
For immunocytochemistry experiments, cells were fixed with 4% PFA for 10 min at RT. Cells fixed on glass coverslips were blocked with 10% donkey serum and 1% BSA (Sigma) in PBS for 30 min at RT, and incubation with primary antibodies was performed in blocking solution overnight at 4 °C. After 3 × 5 min washes in 1X PBS at RT, cells were incubated with Alexa-conjugated fluorescent secondary antibodies (Invitrogen) (1/500, in 1% donkey serum and 1% BSA in PBS) for 1 h at RT. Cells were washed 3 × 5 min in 1X PBS, incubated with DAPI for 5 min at RT and mounted on SuperFrost+ glass slides (Thermo Fisher Scientific).
For immunocytochemistry experiments, cells were fixed with 4% PFA for 10 min at RT. Cells fixed on glass coverslips were blocked with 10% donkey serum and 1% BSA (Sigma) in PBS for 30 min at RT, and incubation with primary antibodies was performed in blocking solution overnight at 4 °C. After 3 × 5 min washes in 1X PBS at RT, cells were incubated with Alexa-conjugated fluorescent secondary antibodies (Invitrogen) (1/500, in 1% donkey serum and 1% BSA in PBS) for 1 h at RT. Cells were washed 3 × 5 min in 1X PBS, incubated with DAPI for 5 min at RT and mounted on SuperFrost+ glass slides (Thermo Fisher Scientific).
Free full text: Click here
